Preparations having a protracted b12 effect and method of preparing same



United States Patent 3,219,532 PREPARATIONS HAVING A PROTRACTED B EFFECT AND METHOD OF PREPARING SAME Knud Morten Kristensen, Birkerod, Denmark, assignor to A/S Dumex (Dumex Ltd.), Copenhagen, Denmark, a Danish company No Drawing. Filed May 17, 1962, Ser. No. 195,395 Claims priority, application Denmark, May 23, 1961, 2,098/ 61 16 Claims. (Cl. 16781) This invention relates to injectable preparations having a protracted vitamin B effect.

It is known that cyanocobalamine and other cobalamines, as for example hydroxycobalamine, are effective in the treatment of pernicious anemia and some other diseases, when applied parenterally.

By intramuscular injection of the said substances, which in the following are comprised by the generic term vitamin B or B only, an increase of the B -contents can be ascertained in the blood serum of the person in question after the injection, said contents, however, decreasing within a few days to the level present before the injection. One reason for this decrease is that it is only possible in a single injection to inject about 30 g. of B without any substantial excretion with the urine taking place, whereas excretion with the urine increases proportionally by increasing doses, so that the excretion amounts to about 80% of the injection dose upon injecting 300 ,ug. in a single dose, and even 95% on injecting 500 pg.

Since the daily dose of B for patients suffering from pernicious anemia in remission is at present evaluated at as high as 10 g, it will be evident from the above that comparatively frequent injections are necessary, and that, accordingly, it is desirable to arrive at preparations having a protracted effect upon injection, that is so-called depot preparations.

For this purpose, suspensions of B in a vegetable oil have been used instead of aqueous solutions. However, the protraction of the effect, which can thus be obtained, only amounts to a few days.

It is further known that vitamin B forms a complex compound with tannin which is slightly soluble in water, but on trying to inject suspensions of the said compound, it has been found that no protracted effect of consequence is obtained. If zinc ions are also present at the precipitation of the B12 tal'l1'1ll'l complex, the said ions also form part of the complex, and a water-insoluble product is obtained, the injection of an aqueous suspension of which results in a lower blood serum level than in the case of an aqueous B -solution, and in a protracted effect which may last for as long as 3 weeks. The excretion with the urine is correspondingly lower.

However, injection of the said preparation produces serious pains at the site of injection, probably because the tannin acts upon the tissue protein, and even if this inconvenience may be reduced to some extent upon addition of a local anesthetic, it cannot be completely removed. Further, a still longer protraction would be desirable.

The present invention relates to preparations, which do not give any inconveniences upon injection, and by means of a single injection of which a satisfying blood serum level can be obtained and maintained for a considerably longer period than hitherto possible.

The said preparations comprise a complex compound of a vitamin B -active substance and tannin suspended in a pharmaceutically acceptable solution of aluminium monostearate in a solvent selected from the group consisting of vegetable oils and liquid fatty acid esters.

It has been found that a suspension of the said kind 3,219,532 Patented Nov. 23, 1965 can be injected intramuscularly without any inconvenience to the patient, and tests have shown that even 10 weeks after a single injection, the concentration of B in the blood serum is substantially above the concentration before the injection. Accordingly, excretion with the urine is quite minimal.

As it appears from the tests hereinafter described, there can hardly be any doubt that the said effect must be due to the character of the suspension medium and probably especially to the gelatinous character of the suspension which is produced by the aluminium monostearate.

It is certainly known, that on suspending procaine penicillin in a vegetable oil, in which aluminium stearate is dis solved, a protracted effect is obtained which lasts for 3-4 days, Whereas a similar aqueous solution gives a therapeutically effective blood level for only about 1 day, but even if thus the conclusion should be drawn that a certain protraction could also be obtained by means of a corresponding preparation of the complex compound of B and tannin, it is, however, surprising and unforeseeable that a protraction can be obtained, which lasts for many weeks, and that large doses may be injected without more than a quite minimal excretion with the urine taking place.

As examples of oils, which may be used as a suspension medium, may be mentioned olive oil, sesame oil and peanut oil. As an example of a usable ester of a fatty acid may be mentioned isopropyl myristate. Generally, an antioxidant, such as cysteine, should be added to the oil to counteract oxidative decomposition of the tannic acid moiety of the complex.

On the lines of the results having been found in the use of aqueous suspensions, the present oil suspensions also show good results when zinc forms part of the complex, and in a preferred embodiment of the present method, therefore, a complex compound of vitamin B zinc and tannin is used according to the invention.

Instead of zinc, other pharmaceutically acceptable metals may form part of the complex, such as bismuth.

It should be noted, however, that incorporation of a metal, for example zinc, in the complex does not result in a greater protraction as observed in aqueous suspensi-ons, so that the protracted effect of the present composition is due to other causes.

It has further been found that by fractioning the tannin forming an article of commerce, a fraction can be isolated which, when used in a smaller amount by weight, gives equal or better precipitation of especially the B zinc-tannin complex than does the product of commerce, so that it is possible to reduce the tannin contents. According to the invention, therefore, a complex compound is preferably used in which the tannin component is a fraction of the said kind produced from tannin, forming an article of commerce.

According to the invention, a particularly simple method of producing the present preparation consists in producing the complex compound by mixing aqueous solutions of the components, whereby the complex compound. forms a precipitate which is filtered off, dried and ground into the suspension medium.

According to the invention, however, it is also possible to proceed in the manner that a gel is first produced by dissolving aluminium monostearate in an organic solvent, the complex vitamin B compound being then stirred into said gel, whereafter the organic solvent is evaporated, and the residue is mixed with the vegetable oil. This gives a more intimate incorporation of the complex compound in the aluminium monostearate because the single particles are coated with a layer of the latter before the suspension in the vegetable oil.

By proceeding in this manner, the antioxidant, such as cysteine, is preferably added to the solution of alu minium monostearate.

The ratio of B to tannin may be from 1:2 to 1:5, preferably 1:4, and the preferred suspension ratio is from 2501000 ,ug. per ml. of vegetable oil.

In the following, the production of the preparations will be illustrated by some examples.

EXAMPL'E 1.-B -TANNATE SUSPENSION Under aseptic conditions and using sterile vessels, 500 mg. of B are dissolved in 100 ml. of sterile water, and the solution is sterile-filtered into a suction flask. The filter is washed with 3 x 5 ml. of sterile water, whereafter a solution of 2000 mg. of tannin and 200 mg. of cysteine in 50 ml. of sterile water is also sterile-filtered into the flask. The filter is then washed with 2 x 10 ml. of sterile 1% aqueous NaCl-solution. After standing in a refrigerator for 30 minutes, the precipitate is removed by filtration, and dried in vacuum over P According to a spectrophotometric determination, the resulting powder contains 0.40 mg. of B per mg.

500 mg. of the said powder and 100 mg. of cysteine are ground with 400 ml. of a 2% aluminum monostearate gel in sesame oil in a sterile mortar. The resulting suspension has a particle size of less than EXAMPLE 2.B -ZINC-TANNATE SUSPENSION Under aseptic conditions, 300 mg. of vitamin B are dissolved in 40 ml. of sterile water in a sterile flask. The solution is sucked through a sterile glass filter into another sterile flask, the filter being washed with 10 ml. of sterile water. Through the same filter, a solution of 1200 mg. of tannin and 600 mg. of cysteine in ml. of sterile water is added. After washing with 10 ml. of sterile water, a solution is added through the filter, consisting of 1200 mg. of zinc acetate in 15 ml. of sterile water, whereafter the filter is washed with 10 ml. of sterile water. The flask is then left for 45 minutes at 5 C., whereafter the precipitate is removed by centrifuging. The removed precipitate is dried in vacuum over P 0 A spectrophotometric determination shows a content of 0.15 mg. of B per mg.

500 mg. of the thus obtained complex compound and 100 mg. of cysteine are ground in a sterile mortar with 150 m1. of a 2% solution of aluminium monostearate in sesame oil. Thereby a suspension is produced, containing 0.5 Mg. of B per ml., and having a particle size below 101w.

EXAMPLE 3 .B -BISMUTHTANNATE SUSPENSION In the manner described in Example 2, solutions of 30 mg. of B in 5 ml. of Water, 80 mg. of tannin and 60 mg. of cysteine in 5 ml. of water, and 10 mg. of bismuthsodium tartrate in 5 ml. of water, respectively, are introduced in the said order into a flask through a glass filter and there is washed with 5 ml. of water after the addition of each of the solutions. The resulting precipitate is isolated by centrifuging. A spectrophotometric determination shows that the liquid, which is centrifuged 015?, contains 0.08 mg. of B per ml. or in all 8% of the initial amount of B After drying in vacuum over P 0 the precipitate is ground with 10 mg. of cysteine in 55 ml. of a 2% solution of aluminium monostearate in sesame oil to form the final suspension.

EXAMPLE 4.B -TANNATE SUSPENSION WITH OR WITHOUT METAL AS PART OF THE COM- PLEX COMPOUND 1 g. of aluminium monostearate is heated in 50 ml. of benzene on a water bath at 50 C. until a limpid gel is formed. In the latter, 250 mg. of a complex vitamin B -compound are stirred, which is produced according to one of the Examples 1-3, together with 50 mg. of cysteine,

subsequent to both compounds having been ground to a fine powder in a mortar. After evaporation of the benzene, the remaining powder is ground into 200 ml. of sterile sesame oil. The whole procedure is carried out under aseptic conditions, and a sterile suspension is obtained which is easy to shake up, if it precipitates.

For determination of the depot effect, comparative tests have been made with injections of the hereinafter mentioned B -preparations which were marked with the Co isotope.

Male rabbits with a body weight of 2.7-3.0 kg. were given intramuscular injections of the various preparations in a leg muscle under narcosis, and the radioactivity at the injection site was kept under observation by means of a scintillation detector. The following preparations were used:

A. Vitamin B in aqueous 0.9% NaCl solution with a content of 500 ,ug. of B per ml. and 0.18 microcurie of B -Co per ml.

B. Vitamin B -zinc-tannate in aqueous B -contents and contents of Co as in A.

C. Vitamin B -zinc tannate suspended in a 2% solution of aluminium stearate in sesame oil with 500 ,ug. of B and 0.5 microcurie of B Co per ml.

D. Vitamin B tannate suspended in the same medium as preparation C, and with the same B and B Co contents per ml. as C.

E. Vitamin B tannate suspended in sesame oil without aluminium monostearate, contents of B and C0 as in C.

From preparations A and B were used 0.30 ml. and from C, D and E 0.25 ml. for each injection.

In the following Table 1, the amount remaining on the injection site at different times after the injection is given in percent of the injected amount. Table 2 gives the percentual amount which has been excreted with the urine during the 1', 2, and 3 day after the injection,

suspension.

respectively.

Table 1 Time interval after the injection Preparation 1 hour 1 day 4 days 10 days 25 days Table 2 Percent of dose excreted with the urine Preparation 1 day 2 day 3 day The tables show that vitamin B is surrendered at a much slower rate from the oil suspensions (preparations C and D) than from the aqueous media (preparations A and C), and that excretion with the urine is quite minimal. A comparison between the values found for prepa rations D and E shows that the aluminium monostearate has a substantial share in the obtained protracted effect.

Intramuscular injection of the preparations F, G, H and J specified hereinafter has further been made in healthy human beings, and the B contents in the blood serum and urine have been followed by means of microbiological determinations with Lactobacillus leichmannz'i. The injections, each of which contained 300 ,ug. of vitamin B were made in the manner that each test person got all 4 preparations successively and with suitable intervals, the injections being made in the order of G, F, H and I, and the results of the microbiological determination for a test person chosen at random appears from the following Tables 3 and 4 where the blood serum level of B is given in pg. per ml., and the excretion with ing to 500 g. of vitamin B The excretion with the urine during the first day was here 0.12 g. of B corresponding to 0.2 pro mille.

The determinations of B in the urine have been made microbiologically with Loctobacillus leichmannii, and in the same manner the blood serum levels of B were determined. The values of the latter, which have been determined until now, is given in the following Table 5 in pg. per ml. For comparative purposes, the blood serum level in healthy persons is between 150 and 800 the urine lS given in ,ug. of B per day. pg. per ml.

Table 5 Blood serum level of B12 in pg. per ml.

Patient No. 1 hour Days after injection alter injection 1 2 3 4 6 8 12 15 10 21 25 34 43 54 Table 3 Blood serum level of Bi in pg. per milliliter lPreptn'ation Before Time intervals after injection in ection lhour ld. 3d. 6d. 9d. 12d. 24d. 48d. 72d.

G 320 2,940 1, 150 450 F 320 10,550 775 350 H 320 1,525 l, 250 800 J 320 580 730 570 430 Table 4 Excreted Bn with urine in the day in question (in g.) Time intervals after injection of preparation F G H I 1 day 2 day 3 day The normal excretion without administration of B is less than 0.02 ,ug. per day.

It appears from Table 3 that on suspension in oil with added aluminium monostearate of the complex compound consisting of B zinc and tannin, a protraction of the B effect is produced which lasts at least 6 times as long as the protraction which is obtained with the known aqueous suspension of the same complex compound.

From Table 4 is seen that practically no B excretion with the urine takes place after injection of a preparation which is produced according to the invention.

Further comparative tests were made upon 3 patients with untreated typical pernicious anemia.

Patient No. 1 got an intramuscular injection of 540 g. of vitamin B in aqueous solution, of which 412 ,ug. or 77% were excreted with the urine during the first day.

Patient No. 2 got an injection of 1 ml., corresponding to 1000 ,ug. of vitamin B of a suspension of B -zinc tannate in oil suspension with aluminium monostearatc. During the first day after the injection 0.4 g. were excreted with the urine, corresponding to 0.4/ of the injected dose.

Patient No. 3 got 1 ml. of a suspension of B -zinc tannate in oil with aluminium monostcarate correspond- It appears from the table that the blood serum level for patient No. 1 having a very high value immediately after the injection, has decreased to under the allowable level in the course of 16 days, whereas patient No. 2 after 19 days and patient No. 3 after 34 days still show blood levels above the average value of above 400 pg. per ml. for normal persons. Patient No. 3, whose blood serum level at present has been followed for 54 days, has still a blood serum level of twice the minimum value for healthy human beings.

Clinical experiments are continuing, and at present about patients are under treatment and are being followed by determinating the blood serum levels of B The results indicate that starting from a level of 50 pg. per ml. or even less, intramuscular injection of a single dose of the composition of the invention with a 13 content corresponding to 1 mg. of cyanocobalamine is sufficient to bring the level up and keep it at or above the normal value in healthy persons of 300-400 pg. per ml. for at least 24 months.

I claim: 5

1. A preparation having a protracted vitamin B effect, comprising a complex compound of a vitamin B -active substance and tannin suspended in a pharmaceutically acceptable solution of aluminium monostearate in a solvent selected from the group consisting of vegetable oils and isopropyl myristate.

2. A preparation having a protracted vitamin B ei iect, comprising a complex compound of a vitamin B -active substance, zinc and tannin suspended in a pharmaceutically acceptable solution of aluminium monostearate in a solvent selected from the group consisting of vegetable oils and isopropyl myristate.

3. The preparation of claim 2, in which an antioxidant is added to the solution of aluminium monostearate.

4. An injectable composition, comprising a suspension of a complex compound of a vitamin B -active substance, zinc and tannin, in a solution of aluminium monostearate in a pharmaceutically acceptable vegetable oil.

5. An injectable preparation having protracted vitamin B efiect, consisting essentially of particles of a complex compound of vitamin B -active substance and tannin suspended in a pharmaceutically acceptable suspension of aluminium monostearate in a liquid selected from the group consisting of vegetable oils and isopropyl myristate, the ratio of vitamin B -active substance to tannin being that resulting from precipitating 1 part by weight of the former with 2 to 5 parts by weight of the latter.

. 6. An injectable composition comprising a suspension of a complex compound of a vitamin B -active substance and tannin, in a solution of aluminium monostearate in a pharmaceutically acceptable vegetable oil.

7 7. An injectable composition of the preparation of claim 5, containing an antioxidant.

, 8. An injectable preparation according to claim 5 in which the suspension ratio is from 250 to 1000 micrograms of the B -active substance per ml. of the suspension liquid.

9. An injectable composition having protracted vitamin B eifect, consisting essentially of particles of cyanocobalamine and tannin, the ratio of the cyanocobalamine to tannin being that obtained by precipitating 1 part by weight of the former with 4 parts by weight of the latter, in a pharmaceutically acceptable suspension of aluminum monostearate in sesame oil, the ratio of aluminum monostearate to oil forming a gel, the ratio of cyanocobalamine and tannin to gel being about 1 to 720 by weight, and the particle size of suspended particles being less than 10 microns.

10. A preparation according to claim 5 in which the ratio of B -active substance to tannin is that resulting from precipitating 1 part by weight of the former to 4 parts by weight of the latter and the oil is sesame oil.

11. A preparation according to claim 5 in which the complex compound is of a vitamin B -active substance, bismuth and tannin.

12. The method comprising preparing a composition having protracted vitamin B effect wherein a vitamin B -active substance and tannin are reacted by admixture in aqueous solution to form a B -active substance con- 8 taining precipitate, separating the precipitate containing active product, and drying the same, and suspending the dry precipitate in a pharmaceutically acceptable solution of aluminum monostearate in a solvent selected from the group consisting of vegetable oils and isopropyl myristate.

13. The method of claim 12 in which the aqueous solution contains zinc acetate to form a complex compound of vitamin B -active substance, zinc and tannin.

14. In the method of preparing compositions having protracted vitamin B elfect which consists essentially in heating aluminium monostearate in benzene until a limpid gel is formed, mixing into the gel a vitamin B tannin complex compound, evaporating off the benzene, grinding the residue to a powder to form a pharmaceutically acceptable suspension in a solvent selected from the group consisting of vegetable oils and isopropyl myristate.

15. In the method of preparing compositions having protracted vitamin B effect which consists essentially in heating aluminium monostearate in benzene until a limpid gel is formed, mixing into the gel a vitamin B zinc tannin complex compound, evaporating ofi the henzene, grinding the residue to a powder to form a pharmaceutically acceptable suspension in a solvent selected from the group consisting of vegetable oils and isopropyl myristate.

16. A preparation according to claim 5, in which the vitamin B active substance is hydroxycobalamine, the ratio of the hydroxycobalamine to tannin is that obtained by precipitating 1 part by weight of the former with 4 parts by weight of the latter, the liquid is sesame oil, the ratio of aluminum monostearate to oil forming a gel, the ratio of hydroxycobalamine tannin to gel is about 500 mg. of hydroxycobalamine tannin to 400 ml. of a 2% aluminum monostearate gel, and the particle size of suspended particles is less than 10 microns.

References Cited by the Examiner UNITED STATES PATENTS 10/1956 Buckwalter 16782.9 1/1960 Thompson "167-8 1 

1. A PREPARATION HAVING A PROTRACTED VITAMIN B12 EFFECT, COMPRISING A COMPLEX COMPOUND OF A VITAMIN B12-ACTIVE SUBSTANCE AND TANNIN SUSPENDED IN A PHARMACEUTICALLY ACCEPTABLE SOLUTION OF ALUMINUM MONOSTEARAE IN A SOLVENT SELECTED FROM THE GROUP CONSISTING OF VEGETABLE OILS AND ISOPROPYL MYRISTATE. 